In order to dissect the biochemical steps involved in genetic recombination we have chosen to focus on a key early step: strand exchange between homologous parental DNAs. In vitro, the product of this strand exchange reaction is a joint molecular composed a single-strand DNA joined to one end of a linear duplex DNA. We have established a new paradigm for this homologous pairing, in essence, that recombinases such as the E. coli recA protein can hybridize a single strand of any sequence and an intact duplex. That is, the three strands form a novel DNA triplex (R-form DNA) in which the third strand may include both purines and pyrimidines. Thermal denaturation of chemically substituted DNAs and chemical footprinting of R- form DNA confirm that the third strand in R-form DNA is in the major groove of the duplex. We have also been able to isolate synaptic complexes consisting of all the three strands and recA. These structures have been studied in detail and will continue to be the basis of additional structural investigations. The kinetics of formation of these complexes are being used as a model for the homology search process in the obligatory recombination events during meiosis. The synaptic complexes have also been used to develop a method for the selective cleavage of human DNA (RecA- Assisted Restriction Endonuclease (RARE) cleavage); this method is now being applied to map and clone large fragments of DNA close to the Huntington Disease and Multiple Endocrine Neoplasia I genes. In addition, we have been successful in cloning the gene for a thermostable recA homologue. This protein should be very useful for mechanistic studies and for several important applications in biotechnology. Finally, we have cloned from yeast and higher eukaryotes several sequence homologues to recA. We are attempting to establish functional homology between the proteins encoded by these genes and E. coli recA protein.